Alkyl-Substituted Hydroxamate Resin for Use in a Generator System

This application claims the benefit of U.S. Provisional Application No. 63/344,194, filed on May 20, 2022, which is incorporated herein by reference in its entirety.

This invention was made with government support under contract number DE-SC0012704 awarded by the U.S. Department of Energy. The United States government has certain rights in the invention.

Scandium 44 (Sc) is of high interest as a potential positron emission tomography (PET) imaging radionuclide due to its promising characteristics such as high positron branching ratio (94.27%) and 3.97 hour half-life, making it a suitable candidate for radiolabeling of small to medium biomolecules since it matches their biological half-lives.Sc has even shown slightly superior image resolution in radiopharmaceutical imaging compared to the analogousGa systems. Presently,Sc can be obtained in one of two ways; irradiation ofCa targets with low energy protons or through decay of the parent isotopeTi in aTi/Sc generator system. While the solid targetry approach on calcium produces high yields ofSc, due to the short half-life ofSc the direct production requires access to a cyclotron at or near the imaging facility. More importantly during the irradiation several other Sc isotopes are produced which then requires extensive post-irradiation processing and purification before theSc can be subsequently used for radiolabeling. Employing a generator system allows for an alternative approach to the production ofSc. In these systems a long-lived parent radionuclide (Ti) is adsorbed onto a solid phase matrix to facilitate chromatographic separations. Once the parent nuclide decays to the short-lived radionuclide of interest it is removed from the solid support through a facile process in a pure form. TheTi/Sc generator system would allow for daily elution ofSc without the need for on-site irradiation, and the long half-life of parent isotopeTi (60 years) would allow for a potentially long-lasting source of pureSc.

Development of an acceptableTi/Sc radionuclide generator needs to address several criteria such as efficient separation, lowTi breakthrough, long term stability and useful Sc eluates that will be suitable for subsequent radiolabeling (low volume, low pH, high purity). Several current generators employ the use of a commercial AG 1-X8 anion exchange resin eluting with dilute oxalic acid/hydrochloric acid mixtures, but there are numerous drawbacks to this method, such as the extremely large volumes needed to elute sufficientSc activity, short generator lifetimes beforeTi breakthrough, and the presence of competing oxalates in solution that will hinder subsequent radiolabeling.

More recently the use of commercially available ZR resin has been suggested as a possible resin for theTi/Sc radionuclide generator system. The Triskem hydroxamate based ZR resin has been previously used to separate Zr from Y, and due to the chemical similarities between the Zr(IV)/Y(III) and Ti(IV)/Sc(III) pair, it also shows high selectivity for Ti and little selectivity for Sc over a range of acid concentrations. While the high retention ofTi on the ZR resin indeed seems promising, there is little to no information in the literature on theSc elution activity or generator lifetime. One study conducted by Radchenko et al. suggestsTi breakthrough after 40 bed volume elutions of a conventional direct elution generator. Using ZR resin generators, the elution volumes needed compared to the AG 1-X8 generators is drastically decreased, but the generator lifetime beforeTi breakthrough as well asSc activity elution and purity of elutedSc has been extremely inconsistent and unreliable. There is an organic impurity that is eluted along with theSc which inhibits subsequent radiolabeling with DOTA, HOPO or NOTA under standard conditions. Thus, to date, there are no commercially available resins that provide a robust generator system that tightly retainsTi while supplying pureSc in a convenient form for radiolabeling without the use of tedious post-elution processing and purification.

Despite advances in radionuclide generator research, there is still a scarcity of generators that retainTi while continuously supplyingSc that is free of organic impurities that can hinder subsequent radiolabeling. Such a system would eliminate the need for post-elution processing and purification and could operate at reduced elution volumes compared to known methods. These needs and other needs are satisfied by the present disclosure.

The present disclosure is directed to a hydroxamate-based resin for use in aTi/Sc generator system. In an aspect, the carboxylate groups of a commercially available resin can be synthetically modified to produce an alkyl-substituted hydroxamate resin of Formula I (V$LG),

The disclosure also relates to aTi/Sc generator system comprising an alkyl-substituted hydroxamate resin of Formula I and a method of producingSc, the method comprising decay ofTi in aTi/Sc generator system using an alkyl-substituted hydroxamate resin of Formula I. In an aspect, the alkyl can be methyl.

The disclosure further relates to aHf/Lu generator system comprising the use of an alkyl-substituted hydroxamate resin of Formula I and a method of producingLu comprising decay ofHf in aHf/Lu generator system using an alkyl-substituted hydroxamate resin. In an aspect, the alkyl can be methyl.

Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.

Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

In one aspect, in the methods disclosed herein, the carboxylate groups of commercially available Waters™ Accell Plus CM resin and other commercially available resins can be synthetically altered to produce alkyl-substituted hydroxamate functional groups, such as, for example, methyl-substituted hydroxamate functional groups. In an alternative aspect, a commercially available hydroxamate resin can be used in the methods disclosed herein. In a further aspect, the silica-based media of the Accell resin allows for a chemically robust resin backbone, and, without wishing to be bound by theory, the alkyl substituent on the hydroxamate functional group lowers the pKof the modified resin, compared to the protonated/unsubstituted form which can allow for stronger metal binding.

In an aspect, R′ represents the alkyl substituent on the hydroxamate functional group. In a further aspect, alkyl groups can branched or unbranched, can be saturated, and can have from 1-12 carbon atoms in their longest chains, or can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Further in this aspect, non-limiting examples of suitable straight-chained, saturated alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl groups and dodecyl. In a preferred embodiment, the straight chain, saturated alkyl group can be a methyl group.

In another aspect, non-limiting examples of suitable branched, saturated alkyl groups include isopropyl, isobutyl, sec-butyl, t-butyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl (isopentyl), 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl (neopentyl), 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl groups, and 2-methyl-5-ethyldecyl. In a preferred embodiment, the branched, saturated alkyl groups include isopropyl and/or t-butyl.

In one aspect, the disclosed alkyl-substituted hydroxamate resin can be synthesized in two steps using commercially available reagents under mild conditions (Scheme 1). Further in this aspect, R is a resin backbone that can be a polymer-coated silica based media. In a still further aspect, RCOOH can be preferably a Waters™ Accell Plus CM resin. In one aspect, R′ is an alkyl group that can be branched or unbranched, can be saturated, and can have from 1-12 carbon atoms in its longest chain.

In a preferred embodiment, R′ is methyl. In one aspect, a methyl-substituted hydroxamate resin can have Formula II, shown below:

In one aspect, a methyl-substituted hydroxamate resin is synthesized as follows. In an aspect, Accell Plus CM resin (1.0 g) is suspended in water (8.0 mL) in a Falcon tube and a solution of 2,3,5,6-tetrafluorophenol (TFP) in acetonitrile (1.0 mL, 1.2 M) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) (0.385 g) are added. In a further aspect, this is allowed to mix by inversion at room temperature for 1 hour after which an additional molar equivalent of TFP solution (1.0 mL, 1.2 M) and EDAC (0.385 g) are added to the Falcon tube. In one aspect, the solution is then mixed by inversion at room temperature for 2 hours. In a further aspect, the resin can then be isolated by vacuum filtration, washed with water and acetonitrile, and dried by continuous suction. In another aspect, the ester resin can then be converted to the N-methyl-substituted hydroxamate resin by reacting with N-methylhydroxylamine hydrochloride (0.837 g) in a methanolic 1 M NaOH solution at room temperature for 18 hours and mixing by inversion. In a still further aspect, the final resin can be isolated by vacuum filtration, washed with water and acetonitrile, and dried by continuous suction. In any of these aspects, the resins can be characterized using ATR-IR spectroscopy.

In any of these aspects, the resin can preferentially bind a parent isotope over a daughter isotope. In an aspect, the resin can have a distribution coefficient of greater than or equal to 5000 for the parent isotope and a distribution coefficient of less than or equal to 5 for the daughter isotope. In one aspect, the parent isotope can beTi and the daughter isotope can beSc. In an alternative aspect, the parent isotope can beHf and the daughter isotope can beLu.

Also disclosed are radionuclide generator systems having an elution bed, wherein the elution bed contains an alkyl-substituted hydroxamate resin as disclosed herein. In a further aspect, the radionuclide generator systems preferentially retain a parent isotope in contact with the alkyl-substituted hydroxamate resin while allowing a daughter isotope to be eluted. In some aspects, the elution bed can have a bed volume of from about 0.3 mL to about 2 mL, or of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2 mL, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values. In another aspect, the radionuclide generator system can be loaded with from about 20 μCi to about 10 mCi of the parent isotope, or about 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 850, 900, or 950 μCi, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mCi of the parent isotope, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values. In an aspect, the parent isotope can decay while in contact with the alkyl-substituted hydroxamate resin, releasing the daughter isotope.

Also disclosed herein is a method for producingSc, the method including contactingTi with the disclosed alkyl-substituted hydroxamate resin, allowing at least a portion of theTi to decay toSc, and eluting theSc. In an aspect, theSc can be eluted with a dilute aqueous solution. In one aspect, the dilute solution can be an acid such as, for example, HCl, or can be saline, or any combination thereof. In another aspect, the HCl can have a concentration of from about 0.01 M to about 10.8 M, or from about 0.5 M to about 8 M, or of about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or about 10.8 M, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values. In one aspect, theSc can be eluted with at least one bed volume of the acid, or can be eluted with two, three, four, or more bed volumes of the acid. In still another aspect, the elution step can be repeated at least once before the resin releases anyTi. In some aspects, depending on bed volume,Ti purity, and other factors, step (c) can be conducted at least 75 times before the resin releases anyTi.

In one aspect, the methyl-substituted hydroxamate resin can be tested in comparison to the commercially available ZR resin in side-by-side generator evaluations. In a further aspect, the robustness of the generator can be investigated by monitoring: the time it takes untilTi breakthrough, and the yield ofSc. Further in this aspect, generators were loaded with a 20 μCiTi sample onto a 300 μL bed volume column in 2 M HCl and eluted daily with four bed volumes of 0.5 M HCl. In one aspect, each eluted fraction was monitored forTi breakthrough and the total amount of elutedSc activity was calculated using high purity germanium (HPGe) γ spectroscopy. In another aspect, onceTi breakthrough was observed the generator was discarded, the elution profile for theSc comparison is shown below (). In a still further aspect, during the 11th elution the ZR generator displayedTi breakthrough, as observed through the detection of the 67.9 and 78.3 keV gamma peaks with an instrumentation error of <10%. The disclosed methyl hydroxamate resin (AM) generator was eluted for a total of 75 elutions before anyTi breakthrough was observed.

In an aspect, the lifetime of the generator using the methyl hydroxamate resin is almost seven times longer than the ZR resin generator, and overall showed increasedSc activity elution under the same conditions. In a further aspect, initial optimization studies also show drastically increasedSc activity elution by varying the concentration of the HCl eluent. In one aspect, on a 300 μL bed volume generator using the disclosed AM resin loaded with 100 μCiTi, only 9%Sc was eluted in 4 bed volumes of 0.5 M HCl, but 86%Sc was eluted in the same volume using 2 M HCl with no observableTi breakthrough.

Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a radionuclide,” “a resin,” or “an eluent,” includes, but is not limited to, mixtures or combinations of two or more such radionuclides, resins, or eluents, and the like.

It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y′, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y′, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a resin refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. achieving the desired level of separation ofTi fromSc over the desired time period. The specific level in terms of wt % in a resin required as an effective amount will depend upon a variety of factors including the amount of resin, chemical identity of the resin including any substituents, initialTi toSc ratio, and eluent identity and concentration.

As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

“Cold” as used herein refers to a reaction or molecule that is not radioactive. In an aspect, reactions and processes can be optimized using cold reagents and materials prior to performing the same reactions and processing with radioactive reagents and/or materials.

Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).

Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

All chemicals were used without further purification. Hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, N-phenylhydroxylamine, 2,3,5,6-tetrafluorophenol (TFP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), potassium hydroxide, SOCl, anhydrous diethylether, acetonitrile (ACN), and HPLC grade methanol (MeOH) were purchased from Sigma Aldrich. Optima grade hydrochloric acid (HCl) was purchased from Fisher Scientific (Pittsburgh, PA, USA) and diluted to suitable concentrations (6 M, 4 M, 2 M, 0.5 M, 0.1 M) with 18 MΩ water (25° C., Milli-Q, Millipore, Burlington, MA, USA). ZR resin was purchased from Triskem Intl. (Brunz, France), Amberlite IRC50 resin and Amberlite CG50 resin were purchased from Sigma Aldrich and Accell Plus CM resin was purchased from Waters (Milford, MA, USA). Deionized Milli-Q water (18 MΩ, Millipore) which had been purified by passing through a 10 cm column of Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) was used in all reactions and solution preparations.

The absolute radioactivity ofSc andTi was measured by γ-spectrometry using a high-purity germanium (HPGe) detector, samples were counted for 10 minutes.Ti was detected directly via the 67.87 keV (93.0%) and 78.32 keV (96.4%) γ-lines andSc was detected via the 1157.02 keV (99.9%) γ-line.

Accell Resins: Ester Resin. Accell Plus CM resin (1.0 g) was suspended in Chelex water (8.0 mL) in a 15 mL Falcon tube. TFP solution (1.0 mL, 1.2 M in ACN, 1.20 mmol) and EDAC (0.385 g, 2.48 mmol) were added to the Falcon tube. The reaction was mixed by inversion at room temperature for 1 hour, after which an additional 1.0 mL of TFP solution (1.2 M in ACN, 1.20 mmol) and EDAC (0.385 g, 2.48 mmol) were added to the reaction to ensure complete conversion of the carboxylate groups to the ester resin. The reaction was mixed by inversion at room temperature for a further 2 hours after which the final resin was isolated by vacuum filtration and washed with 3×15 mL water and 3×15 mL ACN and dried by continuous suction. This resin can be stored dry under ambient conditions without any apparent degradation or hydrolysis. IR (ATR, selected bands, v): 3384, 1781, 1670, 1065, 956, 794, 452 cm.

Accell Resins: UH Resin (AU). Hydroxylamine hydrochloride (0.695 g, 10.0 mmol) was dissolved in 1 M NaOH (1.0 mL) and MeOH (2.0 mL) to form the free base hydroxylamine. The ester functionalized resin (1.00 g) was added to this solution in a 15 mL Falcon tube and was mixed by inversion at room temperature for 18 hours. The hydroxamate resin was then isolated by vacuum filtration and washed with 3×15 mL water and 3×15 mL ACN. This resin can be stored dry under ambient conditions without any apparent degradation or hydrolysis. IR (ATR, selected bands, v): 3347, 1731, 1660, 1451, 1062, 795, 451 cm. Formation of the hydroxamate functional group was also verified by the visual formation of a dark red complex upon addition of Fe(III) in dilute acid to the resin.